University of Nebraska - Lincoln
DigitalCommons@University of Nebraska - Lincoln
US Army Research
US Department of Defense
1-1-2005
Miniaturized Lead Sensor Based on Lead-Specific
DNAzyme in a Nanocapillary Interconnected
Microfluidic Device
In-Hyoung Chang
Joseph J. Tulock
Juewen Liu
Won-Suk Kim
Donald M. Cannon, Jr.
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Chang, In-Hyoung; Tulock, Joseph J.; Liu, Juewen; Kim, Won-Suk; Cannon, Jr., Donald M.; Lu, Yi; Bohn, Paul W.; Sweedler, Jonathan
V.; and Cropek, Donald M., "Miniaturized Lead Sensor Based on Lead-Specific DNAzyme in a Nanocapillary Interconnected
Microfluidic Device" (2005). US Army Research. Paper 8.
http://digitalcommons.unl.edu/usarmyresearch/8
This Article is brought to you for free and open access by the US Department of Defense at DigitalCommons@University of Nebraska - Lincoln. It has
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Authors
In-Hyoung Chang; Joseph J. Tulock; Juewen Liu; Won-Suk Kim; Donald M. Cannon, Jr.; Yi Lu; Paul W.
Bohn; Jonathan V. Sweedler; and Donald M. Cropek
This article is available at DigitalCommons@University of Nebraska - Lincoln: http://digitalcommons.unl.edu/usarmyresearch/8
Environ. Sci. Technol. 2005, 39, 3756-3761
Miniaturized Lead Sensor Based on
Lead-Specific DNAzyme in a
Nanocapillary Interconnected
Microfluidic Device
IN-HYOUNG CHANG,†
JOSEPH J. TULOCK,† JUEWEN LIU,†
WON-SUK KIM,†
DONALD M. CANNON, JR.,† YI LU,†
PAUL W. BOHN,†
JONATHAN V. SWEEDLER,† AND
D O N A L D M . C R O P E K * ,‡
Department of Chemistry and the Beckman Institute for
Advanced Science and Technology, University of Illinois at
Urbana-Champaign, 600 South Mathews Avenue, Urbana,
Illinois 61801, and Construction Engineering Research
Laboratory, U.S. Army Engineer Research and Development
Center, Champaign, Illinois 61822
A miniaturized lead sensor has been developed by
combining a lead-specific DNAzyme with a microfabricated
device containing a network of microfluidic channels
that are fluidically coupled via a nanocapillary array
interconnect. A DNAzyme construct, selective for cleavage
in the presence of Pb2+ and derivatized with fluorophore
(quencher) at the 5′ (3′) end of the substrate and enzyme
strands, respectively, forms a molecular beacon that is
used as the recognition element. The nanocapillary array
membrane interconnect is used to manipulate fluid
flows and deliver the small-volume sample to the beacon
in a spatially confined detection window where the
DNAzyme is interrogated using laser-induced fluorescence
detection. A transformed log plot of the fluorescent
signal exhibits a linear response (r2 ) 0.982) over a Pb2+
concentration range of 0.1-100 µM, and a detection limit of
11 nM. The sensor has been applied to the determination
of Pb2+ in an electroplating sludge reference material,
the result agreeing with the certified value within 4.9%.
Quantitative measurement of Pb2+ in this complex sample
demonstrates the selectivity of this sensor scheme and
points favorably to the application of such technologies to
analysis of environmental samples. The unique combination
of a DNAzyme with a microfluidic-nanofluidic hybrid device
makes it possible to change the DNAzyme to select for
other compounds of interest, and to incorporate multiple
sensing systems within a single device for greater flexibility.
This work represents the initial steps toward creation of
a robust field sensor for lead in groundwater or drinking
water.
Introduction
Bioavailable lead is a toxic element, linked to a variety of
adverse health effects (1-3). Since lead is not biodegradable,
* Corresponding author phone: (217) 373-6737; fax: (217) 3737222; e-mail: Donald.M.Cropek@erdc.usace.army.mil.
† University of Illinois at Urbana-Champaign.
‡ U.S. Army Engineer Research and Development Center.
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ENVIRONMENTAL SCIENCE & TECHNOLOGY / VOL. 39, NO. 10, 2005
it accumulates in the environment and produces toxic effects
in plants and animals, even at low concentrations (4-6).
With growing understanding of the health effects of lead, the
U.S. government has increasingly become involved in
addressing the lead threat, and new regulations have been
created for lead. In the Clean Air Act Amendment of 1990,
the EPA designated Pb2+ as an “air toxic”, meaning that it
may cause serious health and environmental hazards when
present as an airborne pollutant (3-7). Also, new guidance
from the Department of Defense Directive 4715 (8) requires
a high degree of management and monitoring of impact areas
on test firing ranges where lead accumulates due to use of
lead-containing munitions. In addition, lead has been listed
as a pollutant of concern in EPA’s Great Water Program (9)
due to its persistence in the environment, potential for
bioaccumulation, and toxicity to humans and the environment. These examples demonstrate that the focus of lead
monitoring has been extended from high-dose effects for
workers in an industrial environment to potential health and
ecological hazards in the global habitat and ecosystem. It is
therefore essential that sensitive, reliable, and cost-effective
analytical methods are developed for the remote monitoring
of lead.
Miniaturization is currently an important trend in environmental monitoring due to its potential to reduce cost,
provide portability, and increase analysis speed. In recent
years, considerable interest has been focused on the development of miniaturized microfluidic (lab-on-a-chip) systems
(10-13) offering improved analytical performance metrics
such as inducing fast and efficient chemical reactions within
small volumes and low manufacturing costs. A further benefit
of miniaturization is the reduction in reagent and sample
consumption and a subsequent reduction in the quantity of
waste produced. Several approaches have been published
for the separation and simultaneous determination of metal
ions based on microfluidic devices (14-21). For example,
Jacobson et al. have demonstrated a successful separation
of Zn, Cd, and Al with detection limits in the 10-100 ppb
range using a capillary electrophoresis (CE) microchip (16).
Deng and Collins utilized colorimetric detection, and six
heavy metals including Pb2+ were effectively separated and
simultaneously determined on a CE microchip with sub parts
per billion detection limits after preconcentration by solidphase extraction (21).
The use of biosensors for environmental pollution monitoring has been another growing area, as these devices
provide rapid, simple, and reliable determination of pollutants at trace levels. Novel lead-specific biosensors have
been developed (22-27). Lu et al. (22, 26-28) developed a
new biosensor for lead by combining the high selectivity of
a DNAzyme with a molecular beacon strategy to achieve
sensitive and quantitative fluorescent detection of Pb2+ over
a wide concentration range from 10 nM to 10 µM.
This study demonstrates the combination of a Pb2+specific DNAzyme biosensor with a multilevel nanofluidicmicrofluidic hybrid device, in which a nanocapillary array
membrane is used to control motion of picoliter-volume fluid
voxels from the analyte-containing sample stream to the
biosensor compartment. These devices employ a membrane
containing an array of nanocapillaries located between
multilayered microfluidic channels, allowing for the convenient and efficient control of fluids in the device (29-31).
In this paper, methods for adapting this lead-selective
DNAzyme to the nanofluidic device are explored, an optimal
protocol is identified, and the analytical figures of merit
including dynamic range, limit of detection, accuracy, and
10.1021/es040505f CCC: $30.25
This article is a U.S. government work, and is not subject to copyright in the United States.
 2005 American Chemical Society
Published on Web 04/15/2005
FIGURE 1. The sensor is composed of a dual-labeled cleavable
substrate DNA whose 5′ and 3′ ends are labeled with a fluorophore
(FAM) and a quencher (Dabcyl), respectively, and an enzyme strand
whose 3′ end is labeled with a Dabcyl. Initially, the fluorescence
of FAM is quenched because of the close proximity of the Dabcyl.
In the presence of Pb2+, the substrate DNA is cleaved, resulting in
the release of fragments and a concomitant increase in fluorescence.
precision are determined. Finally, the microfluidic/DNAzyme
molecular beacon is successfully applied to analysis of Pb2+
in an electroplating sludge certified reference material.
Experimental Section
Materials. Lactic acid and ammonium hydroxide were
obtained from Fisher Scientific (Fair Lawn, NJ). HEPES (N(2-hydroxyethyl)piperazine-N′-(2-ethanesulfonic acid)), sodium chloride, and sodium hydroxide were purchased from
Aldrich (Milwaukee, WI). Prepolymer and curing agent
(Sylgard 184, Dow Corning Corp., Midland, MI) and polycarbonate nuclear track-etched (PCTE) membrane with a
hydrophilic wetting layer of poly(vinylpyrrolidine) (Osmonics,
Minnetonka, MN) were used in the PDMS (poly(dimethylsiloxane)) chip. These PCTE membranes are 10 µm thick
with 200 nm diameter pores at a pore density of 3 × 108
pores/cm2. The lead stock solutions (1000 mg/L) were
purchased from Fisher Scientific as an atomic absorption
standard solution in 2% HNO3. Working solutions of lower
concentration were prepared by serial dilution of the stock
solution with a background electrolyte (BGE). The BGE (25
mM lactate, 25 mM HEPES, and 50 mM NaCl) was prepared
by dissolving lactic acid, HEPES, and NaCl in deionized water
(18.2 MΩ, Milli-Q UV-plus system, Millipore, Bedford, MA).
The pH of the electrolyte was adjusted to 7 with ammonium
hydroxide. Calibration of the chip sensor was performed using
seven different lead concentrations. All reagents were
analytical grade or higher.
Preparation of DNAzyme. The fluorescently labeled
oligonucleotides were purchased from Integrated DNA
Technology Inc. (Coralville, IA). The design of lead DNAzymes
is described in detail by Lu et al. (28). Briefly, a 3′ end Dabcyl
(4-(4′-dimethylaminophenylazo)benzoic acid) labeled enzyme strand, termed 17E-Dy, and a 5′ end FAM (6-carboxyfluorescein) and 3′ end Dabcyl labeled cleavable DNA
substrate, termed 17DS-FD, were chosen (Figure 1). The
DNAzyme enzyme-substrate complex was prepared with
500 nM concentrations of both 17E-Dy and 17DS-FD for
laser-induced fluorescence (LIF) measurements and 2.5 µM
concentrations of both the enzyme and substrate for
fluorescence microscopy studies. A sample of the enzyme
and substrate was heated at 90 °C for 2 min and slowly cooled
to 5 °C for 2 h to anneal the strands together and create the
complex.
Sample Preparation. The electroplating sludge sample
was purchased from Resource Technology Corp. (Laramie,
WY) and prepared for analysis by USEPA Method 3050B (32).
The sludge sample was thoroughly mixed, dried, and ground
immediately before use. For the digestion, 2.5 mL of
concentrated HNO3 and 10 mL of concentrated HCl were
added to 0.1 g of sample, and the mixture was refluxed for
15 min on a hot plate. The digestate was filtered through
filter paper (Whatman No. 41), and the filtrate was collected
in a volumetric flask. The filter paper and the residue were
both washed with 5 mL of hot HCl. These washings were
FIGURE 2. Schematic of a three-dimensional nanocapillary array
interconnect (NAI) gateable microfluidic device. Both crossed
microfluidic channels are identical with dimensions of 50 µm width,
30 µm depth, and 14 mm length.
collected in the same flask. The filter paper and residue were
removed and placed back in the reflux beaker. A 5 mL portion
of concentrated HCl was added, and the mixture was heated
at 95 ( 5 °C until the filter paper dissolved. The residue was
filtered, and the filtrate was collected in the same flask. The
cover and sides of the reflux beaker were washed with HCl,
and this solution was also added to the flask. A control sample
was prepared by following the entire sample preparation
procedure without sludge. The sample was diluted 1:1 using
concentrated ammonium hydroxide followed by a 1000-fold
dilution with background electrolyte before injection on the
microchip. The lead concentration was determined on the
basis of the actual weight of the dried sludge sample and the
final dilution volumes.
Microfluidic Device and Measurement System. Details
of the channel layout and fabrication of multilevel microfluidic-nanofluidic hybrid architectures have been provided
previously (29). A three-dimensional transport device is
depicted schematically in Figure 2. Two identical channels
(50 µm wide, 30 µm deep, and 14 mm long) were orthogonally
oriented on a PDMS microchip and separated at the
intersection by a nanocapillary array interconnect (NAI), the
PCTE membrane with 200 nm diameter cylindrical pores.
Platinum wires (250 µm diameter, Goodfellow Corp., Berwyn,
PA), mounted into reservoirs at the distal ends of the
microchannels, were used to apply bias voltages. An eightrelay system, designed to switch electrical contacts between
Pt electrodes and high-voltage power supplies (Bertan High
Voltage, Hicksville, NY) for different configurations and
magnitudes of microfluidic manipulation, was computer
controlled via a multifunction data acquisition card (DAQ;
National Instruments Corp., Austin, TX) and Labview software
(National Instruments Corp.).
Fluorescence microscopy was used for signal acquisition
using an inverted Olympus epi-illumination microscope
(Melville, NY). The CCD camera (Javelin Ultrichip Hi Res,
Torrance, CA) output was recorded with a videocassette
recorder and a computer-controlled video capture device
(ATI Technologies, Markham, Ontario, Canada). Fluorescence
was excited with 488 nm radiation from an Ar+ ion laser
(Innova 300, Coherent Inc., Santa Clara, CA), which is very
close to the FAM 492 nm excitation maximum. The laser
light was passed through a set of irises and a neutral density
filter (Newport, Irvine, CA) before reaching a dichroic mirror
(505DCLP, Chroma Technology Corp., Brattleboro, VT). The
excitation light was focused by a 10× objective for a 50 µm
diameter area of interrogation. Fluorescence signals were
collected by the same lens and dichroic mirror assembly and
optically filtered through a 100 µm pinhole and band-pass
filter (HQ525/50m, Chroma Technology Corp.) that permits
passage of the 518 nm FAM emission maximum before being
VOL. 39, NO. 10, 2005 / ENVIRONMENTAL SCIENCE & TECHNOLOGY
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detected by a photomultiplier tube (PMT) (HC124, Hamamatsu Corp., Bridgewater, NJ). Control of the PMT data collection
was achieved through a computer with a Labview program
and DAQ card (National Instruments Corp.). All fluorescence
signals were collected at the intersection of the crossed
channels, i.e., just below the nanocapillary array interconnect
membrane.
Results and Discussion
The goal of this study is to adapt the Pb2+-specific DNAzyme
concept to a nanofluidic reagent delivery and detection
scheme to realize a robust, sensitive, regenerable platform
for sensing of Pb2+. The specific recognition of Pb2+ by the
DNAzyme is a necessary, but not sufficient, condition to
realize a biosensor. For sensing, the necessary elements of
reagent delivery and signal generation and recording must
be added. Achieving these objectives requires several advances over the macroscale homogeneous DNAzyme assays,
including (a) using solutions compatible with electrokinetic
control of fluid motion, (b) accomplishing the molecular
recognition reaction efficiently within a small (<100 pL)
volume, and (c) regenerating the analytical reagent, i.e., active
form of the DNAzyme. After optimization of the composition
and device geometry, the overall system is characterized in
terms of common analytical figures of merit and validated
using a standard reference material.
Pb2+-Specific DNAzyme and Electrolyte Optimization.
The synthesis and characterization of the Pb2+-specific
DNAzyme were described earlier in detail (28). The enzyme
DNA strand is a 33-base oligomer, and the substrate DNA
strand is a 20-base DNA/RNA chimer with a single RNA base
whose specific sequences have been previously determined
(22, 28). Briefly, the molecular beacon is constructed by
labeling the 5′ end of the cleavable substrate with the
fluorophore FAM and the 3′ end with a fluorescence
quencher, Dabcyl, and labeling the 3′ end of the enzyme
strand with Dabcyl as shown in Figure 1. When the substrate
(17DS-FD) is hybridized to the enzyme strand (17E-Dy), the
fluorescence of FAM is quenched by inter- and intramolecular
Dabcyl. The melting temperature of the uncleaved substrate
is designed to be above room temperature (∼34 °C) so that
the substrate will not melt from the enzyme strand in this
hybridized state. On addition of Pb2+ (the right-hand side of
Figure 1), the substrate is cleaved at the RNA base site. Upon
cleavage, the melting temperatures of the shorter parts of
the substrate are designed to be below room temperature so
that it will melt and dissociate from the enzyme strand (28).
Thus, FAM is no longer in close proximity to the Dabcyl
quenchers, and the fluorescence increases with the concentration of Pb2+. In a previous paper, the substrate cleavage
reaction was monitored using fluorescence spectroscopy that
illustrated the excellent sensitivity, dynamic range (quantifiable detection in the range of 10 nM < [Pb2+] < 10 µM), and
selectivity (at least an 80-fold selectivity enhancement over
other divalent metals) for Pb2+ (22).
Optimizing the composition of the background electrolyte
(BGE) is critical, because the composition affects biosensor
performance and detection efficiency. The electrolyte must
allow controllable fluidic transfer of lead-containing analyte
solution and delivery of the hybridized DNA strands
(DNAzyme) and sample to the detection window while also
enabling-high efficiency cleavage of the DNA substrate in
the presence of Pb2+. Future generations of the device shown
in Figure 2 may include four fluidic channels: an injection
channel that has a continually refreshed sample stream, a
separation channel that can act as a capillary electrophoresis
column to isolate the analyte from other matrix components,
the nanofluidic gate PCTE membrane, and a detection
channel containing the DNAzyme. Solution flow along any
channel can be controlled via potential bias application.
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Injection of a sample band onto the separation (source)
channel merely requires the application of the correct bias
along the ends of the injection and separation channels. The
migration rate of the analyte must be characterized to know
its movement along the separation channel and the time
interval when the analyte is present at the NAI. Another bias
change will divert solution flow through the PCTE membrane
and thus introduce the analyte into the detection channel.
The analyte is simultaneously removed from potential matrix
interferents and established in the appropriate detection
electrolyte. Clearly, this proposed device may require one
electrolyte for optimal electrophoretic separation and a
second electrolyte for effective DNAzyme cleavage. Current
research is investigating the ability of the NAI to segregate
these cross-channel solutions. While electrophoretic separation is not occurring on the device described in this paper,
the future goal is to optimize DNAzyme performance while
retaining the possibility of preseparating a metal ion mixture
by capillary ion analysis in the microfluidic device. Therefore,
the lactate system, developed by Fritz et al. (33) for the
separation of metal ions and lanthanides by capillary
electrophoresis, became the starting electrolyte. Using the
12 mM lactate system at pH 4, 5, and 7, Pb2+ was separated
from other divalent metal ions such as Mn2+, Cd2+, Co2+,
Ni2+, and Cu2+ in a laboratory CE (P/ACE, Beckman Coulter
Inc., Fullerton, CA) system equipped with indirect UV
detection. For the CE study, 8 mM 4-methylbenzylamine
was used as a UV reagent, the separation voltage was 30 kV,
and a 75 µm i.d. and 60 cm long capillary was used.
Unfortunately, the DNAzyme was not active in the presence
of Pb2+ in these lactate systems. In an effort to improve the
DNA cleavage reaction performance, 50 mM HEPES with 50
mM NaCl (22) was selected as a potential electrolyte. Addition
of 50 mM NaCl was found to play a critical role in stabilizing
the substrate and enzyme strand hybridization reaction,
resulting in improved sensitivity. But CE separation was not
optimal in the HEPES buffer. Finally, the BGE was optimized
by a combination of lactate and HEPES at 25 mM with 50
mM NaCl. Under this condition, Pb2+ was separated from
other metal ions in the laboratory CE system and the DNA
cleavage reaction was efficient (700% fluorescence enhancement in the presence of 5 µM Pb2+). The use of higher NaCl
concentrations was not pursued to prevent generation of
excess current within the microchannels where Joule heating
degrades performance and can eventually cause bubble
formation.
Transport Control and Measurement. The function of
the microfluidic device is to introduce and reactively mix the
lead-containing sample with the Pb2+-selective DNAzyme.
NAI provides a controllable mechanism for fluidic metering
and rapid mixing (29-31, 34). In essence, the PCTE membrane acts as an electronically gateable valve with voltagecontrolled fluid transport rates, preventing fluid flow between
vertically separated microchannels in the off state, and
electrokinetically driving fluid flow across the membrane
when the proper voltage scheme is applied to the fourterminal device. In the off state, any diffusion of analyte across
the NAI is below our detection limit as shown by the use of
fluorescent probes under reverse bias conditions (29, 30).
The electrical bias pathways for the sequence of on and
off states on the microfluidic device are defined in Figure
3a,b. The source channel (horizontal) was filled with 1 µM
Pb2+ solution in BGE, and the receiving channel (vertical)
was filled with 2.5 µM hybridized DNA in BGE. The sequential
images in Figure 3c-h capture the fluorescence from repeated
injections of Pb2+ at the intersection of the cross-channels.
Figure 3c shows the background fluorescence of the twochannel system. The dotted white lines indicate the position
of the horizontal channel, and fluorescence from the vertical
channel is barely observable. In the on state (Figure 3d,e),
FIGURE 4. Fluorescence enhancement as a function of lead ion
concentration from 0.1, 1, 5, 10, 50, 100, and 200 µM. Error bars
represent (1σ (n ) 4). The inset shows the plot of ln(Imax - I) vs
[Pb2+], demonstrating excellent linearity (r2 ) 0.982) over the
0.1-100 µM range.
FIGURE 3. Electrical bias configurations for fluidic control of lead
and DNAzyme within an NAI/microfluidic device: (a) on state and
(b) off state. Temporal sequence of fluorescence images at the
intersection of the crossed microchannels (c-h). The source channel
(horizontal) was filled with 1 µM Pb2+ in BGE (25 mM lactic acid,
25 mM HEPES, 50 mM NaCl), and the receiving channel (vertical)
was filled with hybridized DNAzyme in BGE. Dashed lines indicate
the position of the horizontal source channel. (c) All reservoirs
were floated. (d) Voltage is applied in the on state, causing injection
of Pb2+ solution. Lead solution is transferred from the source channel,
across the NAI, toward the grounded reservoir. The reaction with
DNAzyme produced fluorescence from cleaved DNA in the receiving
channel. (e) During on state bias, the DNA cleavage reaction reached
equilibrium and a constant fluorescent signal was maintained (the
captured image is ∼40 s after on state bias is applied). The Pb2+
plug continued to move to the ground electrode, and cleaved DNA
moved toward the positive bias. (f) ∼1 s after off state bias is
applied. (g) ∼40 s after off state bias is applied. Cleaved DNA
moved toward the positive bias, and the receiving channel was
flushed with bulk hybridized DNAzyme solution. (h) Repetitive Pb2+
plug injection after switching back to the on state bias.
Pb2+-containing solution is electrostatically driven from the
horizontal channel across the NAI to the vertical channel
containing the DNAzyme. To accomplish this transfer,
positive high voltages were applied to the reservoirs at the
two ends of the source channel and the upper reservoir of
the receiving channel while grounding the bottom reservoir
of the receiving channel. The transfer efficiency of Pb2+ is
proportional to the magnitude of applied bias on either end
of the source channel (Figure 3a) up to 400 V, showing larger
transfer ratios at higher voltages. However, bubble generation
due to electrolysis/heating was observed after several repetitive injections at voltages higher than 400 V, thereby
establishing an upper limit on the voltage applied to any of
the channel arms. With a 200 nm pore diameter NAI, the
bias condition illustrated in Figure 3a causes Pb2+ flow from
both arms of the source channel through the nanocapillary
array toward the bottom reservoir of the receiving channel.
As soon as the on state bias is applied (Figure 3d), the
Pb2+ plug moves through the NAI toward the ground end of
the receiving channel and starts to cleave the substrate DNA,
resulting in a significant fluorescence increase. The cleaved,
fluorescently tagged DNA has a slight negative charge and
FIGURE 5. Typical fluorescence signals during repetitive Pb2+
injection (100 nM in BGE). Detection was performed at the
intersection of crossed channels. Electrical bias configurations
are described for Figure 3a,b. The on state was maintained for 20
s and switched to the off state for 10 s, repetitively.
is thus transported toward the positively biased upper
reservoir, giving rise to fluorescence on both sides of the
channel intersection. The off state switches the positive bias
on the source channel to ground, stopping the injection of
Pb2+ into the receiving channel and flushing the cleaved DNA
strand to the upper reservoir (Figure 3f) until all cleaved
DNA is removed from the viewing image (Figure 3g).
Repetitive injections of Pb2+ solution (Figure 3h) show that
this microfluidic chip can be used repeatedly.
Calibration, Precision, and Detection Limit. Calibration
of the DNA biosensor coupled microfluidic system was
accomplished by measuring fluorescence intensity (DNAzyme
substrate strand cleavage efficiency) as a function of Pb2+
concentration in the range of 100 nM < [Pb2+] < 200 µM. A
plot of fluorescence enhancement vs lead concentration is
shown in Figure 4. At each measurement, electrical bias was
cycled four times between the on and off states. Each datum
in the plot represents the average fluorescence enhancement
of these four trials as a function of Pb2+ concentration, and
the error bars represent (σ. The expression of the best fit for
the plot is described as ln(Imax - I) ) -0.0436[Pb2+] + 4.2072,
where Imax and I are the maximum fluorescence enhancement
(%) and fluorescence enhancement (%) at the lead concentration, respectively. The microfluidic system response has
a linear correlation using the above expression over the 100
nM to 100 µM concentration range with a correlation
coefficient (r2) of 0.98. This range likely encompasses Pb2+
concentration levels for most environmental samples. Repetitive detection of 100 nM Pb2+ is illustrated in Figure 5.
During repetitive injection sequences, signals were reproducible with a coefficient of variation of 3.5% (n ) 5) and the
VOL. 39, NO. 10, 2005 / ENVIRONMENTAL SCIENCE & TECHNOLOGY
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TABLE 1. Certified Metal Content in Electroplating Sludge Reference Material
element
concn,a mg/kg
element
concn, mg/kg
element
concn, mg/kg
Al
Cr
Pb
Hg
Na
692.5 ( 82.5
79.5 ( 14.1
119344.0 ( 27453.0
(1.4)
(1576.2)
Ba
Cu
Mg
Ni
Zn
173.3 ( 23.5
63169.3 ( 2410.0
(80.0)
193.6 ( 15.0
182.6 ( 41.0
Ca
Fe
Mn
Ag
562.7 ( 33.0
2,698.7 ( 814.5
17.5 ( 2.1
56.4 ( 6.3
a Certified and noncertified values (36). Values in parentheses are not certified. Certified values are determined on a dry weight basis. Uncertainties
are 1 standard deviation of the measurement. The uncertainty is obtained from 95% confidence intervals.
FIGURE 6. Fluorescence enhancement as a function of lead ion
concentration spiked in the electroplating sludge sample for
standard addition calibration. Error bars represent (1σ (n ) 4).
baseline consistently returned to a constant level. The
detection limit was evaluated by repetitive injection of a 50
nM Pb2+ standard solution. From the baseline noise during
the off state and the fluorescence intensity of 50 nM Pb2+
during the on state, the detection limit (signal-to-noise ratio
of 3:1) was determined to be 11 nM (2.2 ppb), which is lower
than the 72 nM (15 ppb) action level in drinking water
recommended by the U.S. Environmental Protection Agency
(35). These results demonstrate that the combination of
electrokinetically actuated measurement cycles on a microfluidic device and a Pb2+-selective DNAzyme produces a
device sensitive enough to monitor lead in drinking water or
groundwater.
Determination of Lead in an Electroplating Sludge
Standard Reference Material. To challenge the microfluidic
DNAzyme sensor against a complex matrix, it was used for
Pb2+ determination in an electroplating sludge standard
reference material. The certified metal contents in this
material are shown in Table 1. For this assay, the standard
addition method was used to account for matrix effects. In
this electroplating sludge sample, it was also observed that
copper (at a 2-fold higher molar concentration) partially
quenched the fluorescence of cleaved DNA, resulting in a
systematic error in the quantitative detection of Pb2+. Since
the solubility constant of Pb(OH)2 (Ksp ) 2.5 × 10-16) is 3
orders of magnitude larger than that of Cu(OH)2 (Ksp )
1.6 × 10-19) (37), it was possible to effect quantitative removal
of copper in the sludge digestate as a copper hydroxide
precipitate at the electrolyte pH of g8. We confirmed that
copper ion was removed to an undetectable level using the
laboratory CE instrument. The lead ion, on the other hand,
gave a quantitative recovery at pH 8, and the enzymatic DNA
reaction was even more efficient than at pH 7, showing faster
reaction times in the microfluidic device. Other metal ions
did not interfere in the quantitative determination of lead.
Figure 6 shows the standard addition curve for the
determination of lead in the sludge sample. The sludge
digestate was prepared as described earlier, but four aliquots
were spiked with a 50 mM Pb2+ standard solution to make
final added concentrations of 7, 12, 52, and 102 µM Pb2+,
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producing a five-point calibration including the nonspiked
sludge sample. The experiments were conducted in the same
manner as the calibration using the lead standard in buffer
solution. The calibration plot had a correlation coefficient of
0.9993. The concentration of lead in the standard electroplating sludge reference material was determined to be
[Pb2+] ) 125200 ( 3756 mg/kg, a value within 4.9% of the
certified value of 119344 mg/kg, indicating the potential for
excellent accuracy of this microfluidic/DNAzyme system for
Pb2+ determination.
Although this DNAzyme is selective for Pb2+ compared to
its response for other divalent cations, higher selectivities
are required in some applications. This microfluidic device
will be further developed so that the sample analytes are
separated using on-chip capillary electrophoresis, allowing
user-selectable fractions of the sample flow to be introduced
to the DNAzyme (30, 38). For such a system, the selectivity
for particular ions would be enhanced, since it will be
determined by the product of the ability to separate the
desired metal cation from interfering metal ions and the
selectivity of the DNAzyme molecular recognition agent itself.
The sensitivity and robust nature of the DNAzyme can also
be improved by immobilizing the DNA within the NAI pores,
instead of using it in solution (39). This platform offers the
possibility of incorporating multiple sensing locations in one
device; thus, by incorporating different metal-ion-selective
DNAzymes into a single microfluidic device, multiple species
can be determined simultaneously.
Acknowledgments
This work was supported by the Strategic Environmental
Research and Development Program, the Department of
Energy under Grant FG02 88ER13949, and the Engineering
Research and Development Center Long Term Monitoring
Focus Area.
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Received for review August 26, 2004. Revised manuscript
received February 3, 2005. Accepted March 1, 2005.
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